The method and the apparatus serve in particular to count thrombocytes in a sample of non-lyzed whole blood, that is diluted little or not at all.
Platelets or thrombocytes are blood cells having a diameter of 2 micrometers (μm) to 5 μm without a nucleus and of variable shape. They are present in the blood of a healthy subject at a count of 150,000 to 500,000 per microliter. They are involved in the primary hemostasis process and thus in coagulation. Primary hemostasis serves to stop bleeding due to a capillary lesion, but it needs to be reinforced by coagulation if the damaged blood vessel is of greater caliber. The role of thrombocytes is to clump together after they have been activated and to secrete various substances involved in coagulation such as serotonin, calcium, and adenosine diphosphate that are contained in dense granules of cytoplasm. Platelets thus have a very important role in the coagulation phenomenon. Too great a reduction in the number of platelets gives rise to a risk of hemorrhage (to be envisaged prior to a surgical operation, for example). On the contrary, an increase in their number leads to a risk of thrombosis by forming aggregates of platelets. Platelet disorders may be caused by cirrhosis, renal insufficiency (blood not filtered sufficiently by the kidneys), bone marrow disease, a viral infection, or indeed poisoning (phenylbutazone, phenacetin, acetyl salicylic acid, etc.).
The method commonly used for counting blood cells, in particular thrombocytes, is flow cytometry, even though other alternatives exist, such as centrifuging.
The principle of flow cytometry is to count cells by causing them to travel one by one past a measuring system (optical or electrical). This makes it possible both to count the cells by detecting their passage past the measurement system and also to characterize them (size, conductivity, shape, . . . ). In order to enable the cells to go past one by one, the sample is subjected to very high dilution factors. The two types of flow cytometry are optical cytometry (diffusing light and/or emitting fluorescence) and impedance cytometry. Numerous flow cytometers combine both techniques in order to provide more accurate measurement.
Centrifuging a blood sample in a capillary tube serves to separate the various types of cell depending on their density. The various types of cell are initially marked in specific manner. Red corpuscles go to the bottom of the tube, with plasma on top. Between them there is a zone referred to as the “buffy coat” that contains the platelets and various white corpuscles. A float of appropriate density is used for amplifying the buffy coat so as to improve accuracy. By measuring the thicknesses of the various colored layers, it is possible to deduce the number of particles in each layer and thus the number of each type of cell.
Those methods require relatively large amounts of equipment, consequently they can be implemented only in a specialized laboratory. Furthermore, they require relatively large volumes of blood (at least about 100 microliters (μL)).
A very old method of counting thrombocytes that is practically no longer in use comprises using a hemocytometer. A hemocytometer is a device that is essentially constituted by a fluidic section made using glass with a counting grid etched thereon. A sample of lyzed blood that has been diluted at least 100 times and in which the platelets are marked by a non-specific contrast agent of the Giemsa or Wright dye type is introduced into the hemocytometer, which is then observed in a microscope, thereby enabling cells of interest to be counted manually. That method has been abandoned almost completely because of its poor accuracy and because of the lengthy and difficult work it requires of a specialized operator in order to identify platelets and count them manually.
The article by J. S. Lin et al. “A PC-based imaging system for automated platelet identification”, IEEE Transactions on Biomedical Engineering, Vol. 39, No. 9, September 1992, pp. 990-993 describes a method of identifying platelets adhering to a functionalized substrate under flow conditions similar to those encountered in vivo. That method, which includes fluorescent marking of said platelets and using a computer system for processing images, does not make it possible to determine the concentration of platelets in the blood sample, since only cells that adhere to the functionalized substrate are identified.
Documents US 2002/160523 and WO 02/25280 describe methods of detecting thrombocytes based on microvolume laser scanning cytometry (“MLSC”). These methods comprise the introduction of a diluted blood sample into a capillary tube, the scanning of said tube by a focused laser beam and the detection of a fluorescence signal emitted by marked trhombocytes. They are complex and expensive to implement.